1. Field of the Invention
The present invention concerns materials and methods for suppressing non-specific cross-hybridization between repetitive elements present in the target genome or transcriptome and corresponding repetitive elements in nucleic acid probes, while avoiding incidental hybridization between single copy sequences in the probes and adventitious single copy sequences in suppression DNA. More particularly, the present invention concerns the development and use of probes substantially lacking repetitive sequences along with the development and use of suppressive, synthetic repetitive DNA substantially devoid of single copy elements. Even more particularly, such repetitive DNA comprises repetitive sequences corresponding to moderate to high copy repetitive elements adjacent to single copy elements in one or more representative genomic regions.
2. Description of the Prior Art
Genome-wide analysis of gene expression and locus copy number has been facilitated by microarray and array-based comparative genomic hybridization. Persistent questions regarding reproducibility of these techniques have been raised by cross-validation studies in different laboratories1-5. Strategies to mitigate variability in the results obtained from replicate studies have focused on standardizing technical factors, such as array production, RNA synthesis, labeling, hybridization, scanning, and data analysis6-8. Zakharkin et al9 suggest that biological differences among samples is the largest source of this variability and these other factors contribute to a lesser degree.
When analyzing DNA using a hybridization probe, repetitive sequences in the target DNA typically must be blocked prior to hybridization of the probe to the target, in order to avoid high background hybridizations between repetitive elements in the probe and homologous repetitive elements in the target.
Repetitive sequences occur in multiple copies in the haploid genome. The number of copies can range from two to hundreds of thousands, wherein the Alu family of repetitive DNA are exemplary of the latter numerous variety. The copies of a repeat may be clustered or interspersed throughout the genome. Repeats may be clustered in one or more locations in the genome, for example, repetitive sequences occurring near the centromeres of each chromosome, and variable number tandem repeats (VNTRs) Nakamura et al, Science, 235: 1616 (1987); or the repeats may be distributed by Bardoni et al., Cytogenet. Cell Genet., 46: 575 (1987); or the repeats may be distributed over all the chromosomes, for example, the Alu family of repetitive sequences.
Simple repeats of low complexity can be found within genes but are more commonly found in non-coding genomic sequences. Such repeated elements consist of mono-, di-, tri-, tetra-, or penta-nucleotide core sequence elements arrayed in tandem units. Often the number of tandem units comprising these repeated sequences varies at the identical locations among genomes from different individuals. These repetitive elements can be found by searching for consecutive runs of the core sequence elements in genomic sequences.
Competition hybridization, also known as suppression hybridization, provides a means for blocking a potentially overwhelming repetitive DNA signal. The unlabeled competitor or suppressor DNA contains high incidents of repetitive elements which bind to homologous repetitive elements in the target, thereby preventing repetitive portions of the labeled probes from binding to such repetitive elements in the target and increasing the likelihood that the probes will hybridize substantially to the targeted, typically non-repetitive, sequence.
The use of repetitive sequence-enriched (Cot-1) DNA to suppress or block non-specific cross hybridization between repetitive elements present in the probe with other locations in the genome (or transcriptome) is a common requirement for most microarray hybridization studies. Hybridization of suppressor DNA such as Cot-1 to target DNA prior to FISH is commonly practiced in the prior art to avoid background, i.e. non-specific, hybridization. In humans, the Cot-1 fraction is highly concentrated in families of interspersed repetitive elements, such as short and long interspersed repetitive elements, SINEs and LINEs10, 11. Commercial procedures for Cot-1 DNA preparation iterate denaturation and re-annealing of genomic DNA, and are monitored by enrichment for Alu elements (three-fold excess over the corresponding level in the normal genome) and L1 elements (four-fold excess over the corresponding level in the normal genome). Current quality control procedures do not determine the precise composition or sequence of Cot-1 DNA.
While the Cot-1 fraction appears to suppress non-specific hybridization between the repetitive elements of the probe and corresponding or homologous repetitive elements of the target DNA, it also increases experimental noise12 (FIG. 1). Therefore, it was investigated whether differences in Cot-1 composition could be a major source of variability in results from genomic hybridization studies. The role of Cot-1 in genomic hybridization was elucidated by quantitative microsphere by hybridization (QMH)13,20 using sequence-defined, genomic single copy (sc) probes14 and probes composed of contiguous sc and repetitive genomic sequences. It was determined that Cot-1 promotes the formation of stable duplexes (single copy sequences in the probe sequence hybridized to the single copy sequences within the Cot-1 DNA) containing adjacent paralogous repetitive sequences often unrelated to the probe, thereby preventing accurate quantification of single copy sequence hybridization. Incidents of single copy elements within the Cot-1 hybridized to homologous single copy elements in the probe, distort (falsely amplify) the probe signal.
FIG. 1 illustrates hybridization of Cot-1 105 to a genomic DNA target 100 to suppress or block a repetitive element 115 in the target 100 from being available for hybridization with paralogous repetitive elements 120 in the probes 110. Repetitive element 115 is shown in parallel relation to the suppressing repetitive element 117 in the Cot-1 DNA 105, thereby indicating hybridization of the elements 115 to 117. As is typical in the prior art, probes 100 include both single copy elements 135 as well as adventitious repetitive elements 120, the single copy elements 135 being selected and synthesized to selectively hybridize to homologous single copy elements 140 in the target 100. As illustrated, the probes 110 are conjugated to microspheres 145 used for probe 110 detection and quantitation. Probe 110′ is shown hybridized to the target 100, as anticipated by the study design. In addition to hybridizing to the repetitive element in the target 115, however, single copy elements 130 in the Cot-1 105 also hybridize to homologous single copy elements 135 in the probe 125, thereby increasing the probe signal by three fold.
Patent application PCT/US2006/032693 entitled “Quantification of Microsphere Suspension Hybridization and Uses Thereof”, filed Aug. 16, 2006, describes a microsphere suspension hybridization assay utilizing low or single copy genomic hybridization probes allowing direct analysis of whole genomic DNA or RNA) using flow cytometry and is hereby incorporated by reference.
Accordingly, what is needed in the art are methods of suppressing signal distortion caused by hybridization of nucleic acid probes to elements present in Cot-1 DNA, methods of suppressing non-specific hybridization of probes to target DNA; methods of suppressing hybridization of suppressing or competitive DNA to single copy sequences in the target as well as in the probes; methods of identifying and synthesizing suppressive, repetitive DNA; synthesized, repetitive DNA products efficacious for use as suppressor DNA; and nucleic acid hybridization systems utilizing such synthesized suppressive DNA in combination with single copy probes substantially devoid of repetitive elements.